Summary Since its discovery in 1969, Lassa virus (LASV), a bi-segmented RNA virus from the family Arenaviridae, has been recognized as the cause of disease affecting a quarter million people per year, resulting in ~5,000 deaths annually in West Africa. Due to global travel, there have been numerous confirmed cases of LASV infection outside West Africa including the US. Its ability to spread as an aerosol and a case fatality rate of ~15% make LASV a major threat to human health and a BSL 4 pathogen. Unfortunately, no FDA-approved drugs or vaccines are available for treatment of LASV. The overall goal of this project is to address this medical need by identifying and validating small molecule inhibitors of LASV infection as prophylactics and/or therapeutics. The strategy of this project is to identify small molecule inhibitors that target the cellular entry of LASV. Being the first step in viral infection and occurring in the extracellular/endosomal environment, viral entry is a susceptible and accessible target for antiviral therapy. The approach is to leverage our experience with a homogeneous, biochemical, high-throughput screening (HTS) method, AlphaLISA, to identify small molecules that prevent interactions between the glycoprotein of LASV and its host receptor LAMP1. Previously, we developed and applied a biochemical HTS based on AlphaLISA technology to identify compounds that block the Ebola glycoprotein (GP) binding to its host receptor NPC1. Two distinct scaffolds were identified and one exhibited potency against infectious Ebola virus in a murine in vivo study. In Phase I, for Aim 1, an AlphaLISA HTS will be developed and optimized for the identification of small molecules that inhibit the interaction between LASV GP1 and its receptor LAMP1. In Aim 2, a biolayer interferometry (BLI) biochemical assay and cell-based secondary assays utilizing recombinant vesicular stomatitis virus (VSV) carrying arenavirus glcyoproteins (GP) in place of VSV-GP will be built and optimized to confirm initial hits from the primary screen, to determine which interacting partner they bind, and to approximate the affinity of that interaction. In Aim 3, the HTS will be applied to diverse chemical libraries, and hits will be confirmed in the secondary assays. In Aim 4, hits will be validated in infectious Lassa virus assays and prioritized by drug-like structural features and in vitro ADME properties. Together, these assays will identify and validate compounds that suppress LASV infection by inhibiting viral entry and will provide valuable information for prioritizing those inhibitors. In Phase II, we will chemically optimize priority inhibitors for potency and selectivity and evaluate them in animal infection models.